Disorders of inorganic phosphate (Pi) concentration and impairment in Pi reabsorption are common clinical problems. Aging, diabetes mellitus, malignancy, renal failure, alcoholism, transplantation, AIDS, and several therapeutic drugs are well known to cause or to be associated with hypophosphatemia or hyperphosphatemia, mainly by affecting renal tubular Pi transport. The kidney plays a critical role in the regulation of Pi homeostasis. The evidence to date indicates that the majority of renal tubular Pi regulation by dietary and hormonal factors is mediated by the type Ha renal sodium phosphate cotransporter (NaPilla). These factors alter NaPilla surface abundance and thus activity by altering the cotransporter's insertion into and retrieval from brush border membrane (BBM) microvilli. Relatively little is known, however, about the precise events governing NaPilla trafficking to and from the cell membrane. These studies have been limited by the inability to visualize protein trafficking in microvilli with high resolution in real time. Our lab has developed a novel application of total internal reflection fluorescence microscopy (TIR-FM) which allows visualization of trafficking events in brush border microvilli in real time. The proposed studies will use TIR-FM microscopy coupled with fluorescence recovery after photobleaching (FRAP) and image correlation spectroscopy (ICS) techniques to examine how alterations in extracellular Pi and PTH modulate NaPilla trafficking in BBM microvilli. These studies will be performed in opposssum kidney (OK) cells, a well-established model of the proximal tubule. NaPilla trafficking is also belived to be regulated by PDZ proteins (scaffolding proteins that link the cotransporter to the cytoskeleton) and by the cytoskeleton itself. The proposed work will investigate modulation of NaPilla trafficking in BBM microvilli in response to alterations in extracellular Pi or PTH by PDZ proteins and by the cytoskeleton. This work will use dynamic imaging techniques to study physiologic processes at the single protein level in real time. These techniques, besides being widely applicable to other processes, will allow insights into the renal regulation of Pi homeostasis which is crucial for normal cellular function. This knowledge may ultimately lead to the development of novel therapies to treat phosphate imbalance. [unreadable] [unreadable] [unreadable]